Interactions between bioparticles (bacterium, organelle, cell, virus) occurring throughout biology and the adsorption of cells to surfaces in affinity-based separations have a polyvalent nature. Poly- or multivalent interactions are characterized by the simultaneous binding of multiple receptors on the surface of bioparticle to multiple ligands on another surface and can be collectively much stronger than corresponding monovalent interactions.
The difficulty of disrupting multivalent interactions is one of the main problems in designing affinity techniques for cell separation. Theoretical studies have shown that for situations where the number of interactions is >10 it is unlikely that reasonable concentrations of a soluble monovalent competitor (biospecific eluent) can displace the binding equilibrium. Under typical chromatographic conditions (1010-1012 of ligands and receptors per cm2 and 10−10-10−8 cm2 of contact area) the number of specific binding interactions can be between 1 and 10,000. Thus, in most cases an external force is required to simultaneously disrupt multiple bonds and detach specifically adsorbed cells. While leading to cooperative effect, retention of cells on affinity surface correlates with the affinity of individual receptor-ligand binding and the concentration of surface ligand and is sensitive to the presence of competitive binding inhibitors that reduce the force needed to remove attached cells. In approaches currently used for cell release the detachment forces are generated by the passage of air-liquid interfaces (X. Cao, R. Eisenthal, J. Hubble, Detachment strategies for affinity-adsorbed cells, Enzyme. Microbial. Technol. 31 (2002) 153-160; C. Gomez-Suarez, H. J. Busscher, H. C. Van der Mei, Analysis of bacterial detachment from substratum surfaces by the passage of air-liquid interfaces, Appl. Environ. Microbiol. 67 (2001) 2531-2537) or by using flow-induced shear forces (F. Ming, W. J. D. Whish, J. Hubble, Estimation of parameters for cell-surface interactions: Maximum binding force and detachment constant, Enzym. Microb. Technol. 22 (1998) 94-99; C. Cozens-Roberts, J. A. Quinn, D. A. Lauffenburger, Receptor mediated cell attachment and detachment kinetics. II. Experiments model studies with the radial flow detachment assay, Biophys. J. 58 (1990) 857-872). The latter leads to a high degree of dilution of eluted cells and involves the risk of cell damage.
Due to high heterogeneity of cell surface there can be other factors (e.g. hydrophobic and electrostatic interactions, van der Waals attraction) along with affinity interactions that control cell behavior at cell-surface interfaces and must be taken into account when designing affinity adsorbent for cell separations. Until recently, studies of cell adhesion have been focused mainly on cellular response to surface chemistry and topography, on microbial adherence to stiff supports such as polystyrene, teflon and glass. In biological systems cells often come into contact with soft surfaces, e.g. tissues or extracellular matrix that can undergo changes in elasticity (e.g. wound healing). However, only recently systematic studies of the effect of substrate mechanics on cell adhesion has been carried out and softness and elasticity of the surface were shown to be important parameters modulating cell-surface interactions (M. T. Madigan, J. M. Martinko, J. Parker, Brock Biology of Microorganisms. 9-th ed. Upper Saddle River, N.J., USA: Prentice-Hall, Inc., 2000; R. J. Pelham, Y. L. Wang, Cell locomotion and focal adhesions are regulated by substrate flexibility, Proc. Natl. Acad. Sci. USA 94 (1997) 13661-13665; J. Y. Wong, J. B. Leach, X. Q. Brown, Balance of chemistry, topography, and mechanics at the cell-biomaterial interface: Issues and challenges for assessing the role of substrate mechanics on cell response, Surf. Sci. 570 (2004) 119-133.). For example, investigations of the relationship between different types of cells and elasticity of polyacrylamide and alginate-based surfaces have revealed some common changes in cell behavior following a decrease of substrate stiffness, i.e. reduction of a cell spreading and weakening of cell-surface interactions (R. J. Pelham, Y. L. Wang, Cell locomotion and focal adhesions are regulated by substrate flexibility, Proc. Natl. Acad. Sci. USA 94 (1997) 13661-13665; A. Engler, L. Bacakova, C. Newman, A. Hategan, M. Griffin, D. Discher, Substrate compliance versus ligand density in cell on gel responses, Biophys. J. 86 (2004) 617-628; N. G. Genes, J. A. Rowley, D. J. Mooney, L. J. Bonassar. Effect of substrate mechanics on chondrocyte adhesion to modified alginate surfaces, Arch. Biochem. Biophys. 422 (2004) 161-167). Interestingly, these trends are independent of the adhesive ligand. An important implication of such dependence of cell behavior on the mechanics of adsorbent is that the use of soft materials in cell affinity separations may help to avoid or to decrease non-specific cell-surface interactions.
Polyacrylamide-based cryogel monoliths have recently been developed for the applications in bioseparations [V. I. Lozinsky, F. M. Plieva, I. Yu. Galaev, B. Mattiasson, The potential of polymeric cryogels in bioseparation, Bioseparation 10 (2002) 163-188. I. Yu. Galaev, M. B. Dainiak, F. M. Plieva, R. Hatti-Kaul, B. Mattiasson, High throughput screening of particulate-containing samples using supermacro-porous elastic monoliths in microtiter (multiwell) plate format, J. Chromatogr. A 1065 (2005) 169-175.] and are characterized by high porosity and elasticity. Due to the size (10-100 μm) and interconnected structure of the pores and the absence of non-specific interactions with the adsorbent cells pass freely through plain cryogels without affinity ligands [P. Arvidsson, F. M. Plieva, I. N. Savina, V. I. Lozinsky, S. Fexby, L. Bulow, I. Yu. Galaev, B. Mattiasson, Chromatography of microbial cells using continuous supermacroporous affinity and ion-exchange columns, J. Chromatogr. A 977 (2002) 27-38; A. Kumar, F. M. Plieva, I. Yu. Galaev, B. Mattiasson, Affinity fractionation of lymphocytes using supermacro-porous monolithic cryogel, J. Immunol. Methods 283 (2003) 185-194]. Unlike traditional polyacrylamide gels that are rather brittle, polyacrylamide-based cryogels are elastic soft sponge-like materials that can be easily compressed without being mechanically damaged. Due to capillary forces the monoliths retain the liquid inside them and are drainage-protected. As a consequence thereof the effectiveness (calculated as recovered cells in % of totally adsorbed cells) in the liberation of material adsorbed to a polyacrylamide-based cryogel carrying on its surface a ligand capable of interacting specifically or non-specifically with a receptor on the surface of a bioparticle to be separated by elution was found by the present inventors to be low even when applying flow-induced shear forces in order to increase the effectiveness of the elution step.
For that reason the object of the present invention is to provide a process which enables an improved effectiveness in the liberation of adsorbed material to be obtained when eluting bioparticles adsorbed to a macroporous cryogel monolith.